Caenorhabditis elegans (C. elegans) in comparison to single exposures. Endpoints such as germ cell apoptosis, the number of oocytes, brood size, and the life span were employed to evaluate the combined effects of Cd and As on exposed C. elegans from L3 or L4 stages. Our results showed that concurrent exposure to non-toxic concentrations of Cd and As caused the synergy of reproductive and developmental toxicity. The presence of Cd promoted the accumulation of As in both germline and intestine detected by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Although a conversion of As(III) to As(V) was detected as dependent on pH according to the microenvironment of the intestine in the worm, there was no significant difference of toxicity in C. elegans concurrently exposed to Cd and different As species. Using loss-of-function mutant strains, As was deemed responsible for the enhanced joint toxicity, and in which gcs-1 played a key protective role. These data help to better evaluate the comprehensive adverse effects of concurrent exposure of heavy metals at low concentrations on living organisms in the environment.Cadmium (Cd) and arsenic (As) are widely distributed pollutants that co-exist in the environment; however, their joint toxicity on living organisms is still largely unknown. In this study, we explored the joint toxicity of concurrent exposure to Cd and different As species at low concentrations on  Strains including N2 (wild-type Bristol), mtl-2 (gk125), and gcs-1 (ok436) are provided by Caenorhabditis Genetics Center (CGC). Synchronized worms can be obtained when gravid hermaphrodites were lysed in an alkaline hypochlorite solution, and the harvested eggs are hatched overnight at 20 °C without food to remain at L1 stage.According to the standard procedure described by Brenner , all worC. elegans at L4 stage were exposed to designated working solutions on Costar 24-well plates. After exposure, the solutions containing C. elegans were aspirated into centrifuge tubes, and the worms were collected after natural settling, then placed in 400 μL of 75 g/mL AO with OP50 for an incubation at 20 °C for 60 min. After repeated elution, C. elegans were moved to NGM plates for a 60 min recovery on bacterial lawns. The purpose of recovery is to remove AO from the intestine of the worms and make it easier for subsequent detection of apoptotic germ cells. The worms were then anaesthetized with sodium azide 2% (NaN3)  and examined under an Axioscope 5 fluorescence microscope  at 20×. After AO staining, the apoptotic cells were yellow-green, indicating increased DNA fragmentation, while all intact cells were uniformly green. Numbers of apoptotic cells were obtained from at least 20 worms for each group.Germ cell apoptosis: According to the standard procedure described by Gartner , apoptotC. elegans oocyte count was referred to the method of Ruan et al. [C. elegans from each group were placed on agar plate and anaesthetized with a drop of 2% NaN3. The number of oocytes was counted from the spermatheca to the gonad loop per gonad arm under an optical microscope.Oocyte assay: The n et al. . FollowiC. elegans were transferred to new NGM plates every 24 h until oviposition ceased (approximately 5 days), and eggs laid on the NGM plates were counted under a microscope .Brood-size assay: According to the method of Ruan et al. , 15 wormC. elegans grown synchronously to L3 stage were transferred to the plates, and one worm was placed in each well. The C. elegans were observed daily under a microscope, and worms with a lack of response to physical stimulation were deemed dead. Survival curves were recorded by continuously exposing C. elegans to test solutions until all worms were dead.Life-span assay: According to the method of Du et al. , lifespaC. elegans were placed on NGM plates for a cultivation of 48 h and collected. Since the working concentrations were low, we needed to raise a large number of C. elegans for the test. After being exposed in 9 mm Petri dishes for 24 h, C. elegans were washed at least 3 times with Milli-Q water in order to remove the exposure solutions on the nematodes. After washing and removing the supernatant, the worms were frozen at −20 °C in a refrigerator and freeze-dried for at least 48 h until constant weight was reached. Dried worms were precisely weighted, then digested in HNO3  by electric heating at 150 °C. The contents of Cd and As in C. elegans were determined by inductively coupled plasma-mass spectrometry  and atomic fluorescence spectrometer , respectively, based on dry weight.Synchronized L1 C. elegans was determine by LA-ICP-MS at the Mineral Deposit and Exploration Center (ODEC) of Hefei University of Technology, using a laser ablation system (LA) , coupled to Quadrupole-based ICP-MS . L4 worms were treated for 24 h and rinsed three times with Milli-Q water. Randomly selected worms were picked to droplets of Milli-Q water on microscopic slides. Single separated C. elegans were obtained after the droplets have been air-dried. The slides were then loaded onto the LA system according to the method of Dai et al. [The distribution of Cd and As in the i et al. , for a sC. elegans. After pH adjustment, KM solutions of different pH were stocked in centrifuge tubes and marked. Cadmium, As(III), and As(V) at 1000 μg/mL of 4 μL, respectively, were added to 10 mL KM of different pH and mixed for 24 h. Arsenic species change was detected by atomic fluorescence spectroscopy (LC-AFS)  at Instruments Center for Physical Science, University of Science and Technology of China. The carrier gas flow rate was set at 400 mL/min and the shielding gas flow rate at 600 mL/min. The results were processed by LC-AFS Data Workstation V5.5.3.We adjusted the pH of KM by HCl and NaOH  to 3.6, 4, 5, and 6 to simulate the microenvironment in the intestine of p < 0.05 is considered significant.All data of toxicity assays in this study are expressed as mean ± standard deviation, based on at least three independent experiments. The difference between groups is analyzed by One-Way ANOVA with Graphpad prism 8, and C. elegans to 16.4 ± 0.30 days for Cd-As(III), 15.9 ± 0.43 days for Cd-As(V), and 15.8 ± 1.06 days for Cd-As(III)-As(V) from 20 ± 0.50 days for the control group, in agreement with reproductive toxicity , and As(V) are determined based on germ cell apoptosis. As shown in toxicity . These dC. elegans using ICP-MS. Since the non-toxic concentrations were too low to carry out measurements on As species, As accumulation was only determined as total As concentrations in Cd-As(III) and Cd-As(V) groups. The results showed that the mean value of As in the single As(III) group was 0.7 ± 0.15 μg/g, and the mean value of As in the Cd-As(III) group was 0.84 ± 0.04 μg/g, about 1.11 times more than the single exposure group. Similarly, As concentration was 310 ± 29.1 μg/g in the single As(V) group, and the mean value of As was 370 ± 31.2 μg/g when Cd and As(V) were co-exposed, which was 1.17 times more than the single exposure group. Cd content in the C. elegans did not show significant change in single and concurrent exposure groups. LA-ICP-MS was further employed to explore the distribution of Cd and As in C. elegans. Since the non-toxic concentration of As(III) was too low for LA-ICP-MS analysis, the distribution of Cd and As in C. elegans was only measured in the Cd-As(V) group  exposure was 2017.53 and the value of Cd-As(V) co-exposure was 2739.47, in agreement with ICP-MS results.To explore the mechanism behind the synergies of Cd and As, we analyzed the accumulation of the heavy metals in V) group . RandomlC. elegans due to the microenvironment. Considering the complexity of the intestinal microenvironment, we chose pH here to simulate C. elegans intestine, hoping to detect the physicochemical changes of As. The C. elegans gut has a pH range from 6 in the anterior pharynx to 3.6 in the posterior gut [Since As has multiple valence states with different toxicity, ingested As may alter in species in the intestine of rior gut . We adjumlt-2 (gk125) and gcs-1 (ok436) to the same working solutions. mlt-2 (gk125) showed no significant change in the amount of germ cell apoptosis with N2, while gcs-1 (ok436) exhibited more germ cell apoptosis in both single and co-exposure groups of As  and ok436 mutant strain (gcs-1 gene defective type). The main physiological function of metallothionein is involved in detoxification of heavy metals in organisms, including Cd [mtl-2 in C. elegans also plays an important role in protecting against toxic effects caused by Cd detoxification [C. elegans. Cadmium exposure causes inflammatory responses and cellular damage in the human intestine, dysbiosis of intestinal flora, and damage to the intestinal tract [pcs-1 (mt1748) exhibited a higher amount of apoptotic germ cells compared to N2 when exposed to the same level of Cd, indicating that the pcs-1 gene is involved in the toxic regulation of Cd in C. elegans. No difference was found in the amount of germ cell apoptosis between mtl-2 (gk125) and N2, and this is likely due to the similar Cd accumulation at a non-toxic level in single and co-exposure with As. It also suggests that even enhanced As accumulation does not respond to mtl-2. Wang et al. [gcs-1 gene is essential for GSH synthesis in C. elegans cells, so we used the gcs-1 gene defective phenotype to study the toxicity caused by As after co-exposure. It was found that when gcs-1 (ok436) was exposed to Cd alone, there was no significant change in the amount of germ cell apoptosis, showing that non-toxic concentration of Cd does not respond to the gcs-1 gene. After exposure to As(III) or As(V) alone, the amount of germ cell apoptosis in gcs-1 (ok436) increased significantly compared to N2, indicating when gcs-1 was defective in vivo, the worm will be unable to synthesize GSH to protect against the toxic effects caused by As, resulting in increased toxicity. Thus, we believe the higher joint toxicity of Cd-As co-exposure in gcs-1 (ok436) resulted from the increased accumulation of As in vivo. Therefore, the synergistic effect of Cd-As co-exposure was mainly caused by the increased toxicity of As  to As(V) along the digestive tract, but was not enough to cause a significant difference in joint toxicity. We found that germ cell apoptosis in the mtl-2 (gk125) mutant strain was not significantly different from N2, while gcs-1 (ok439) showed higher reproductive damage, confirming that increased As toxicity dominated the synergistic effect of Cd and As. However, due to the low concentration of exposed As(III) and lack of extraction method, As(III) and As(V) cannot be distinguished concurrently in C. elegans, forcing us to rely on the simulation based on the microenvironment of the worm’s intestine. We plan to develop a proper extraction method for As species especially for biological samples, or introduce other in situ detection means such as μ-XANES to address the possible transformation of As(III) and As(V) in vivo. In addition, although the roles of Cd and As in the joint toxicity were determined based on mutant strain studies, the underlying toxicological mechanisms were not fully elucidated. Further exploration focusing on the molecular mechanisms will be carried out in the future. This study indicates co-exposure to heavy metals at non-toxic levels is a potential threat to the ecosystem, which needs further evaluation of other heavy metal combinations and systematical research on the mechanisms involved.Our results showed a synergistic effect of Cd and As at non-toxic concentrations on the reproduction and development of


One anastomosis gastric bypass (OAGB) is an effective and safe treatment for morbidly obese patients. Longer biliopancreatic (BP) limb length is suggested to result in better weight loss outcomes, but to date, no data are available for the OAGB to substantiate this. We hypothesized that applying a longer BP-limb length in the higher BMI classes would result in more weight reduction so that the attained BMI would be comparable to patients with a lower BMI, thereby compensating for differences in baseline BMI.A retrospective cohort study in patients who underwent a primary OAGB at a teaching hospital in the Netherlands between January 2015 and December 2016. BP-limb length was tailored based on preoperative BMI. Patients were divided into three different groups depending on the length of the BP-limb: 150, 180, and 200 cm. Weight loss outcomes after 1 and 3 years and resolution of comorbidities were compared between these groups.Of the 632 included patients, a BP-limb length of 150 cm was used in 172 (27.2%), 180 cm in 388 (61.4%), and 200 cm in 72 (11.4%) patients. Despite more BMI loss, %EWL was lower and attained BMI remained higher in the groups with longer BP-limb lengths. After adjustment for the confounder preoperative BMI, longer BP-limb lengths were not associated with higher BMI loss. There was no difference in remission rates of comorbidities.Attained BMI remained higher in spite of tailoring BP-limb length according to baseline BMI with no differences in remission rates of comorbidities. The Roux-en-Y gastric bypass (RYGB) is considered the gold standard procedure in bariatric surgery for the treatment of morbid obesity . There iIn 1997, Rutledge introduced the one anastomosis gastric bypass (OAGB). Compared to the RYGB, the OAGB has one anastomosis instead of two, which translates into technical ease, shorter operation time, and lower complication rates , 7. FurtSeveral years ago, we made it practice to adjust BP-limb length based on preoperative BMI in our hospital. Our hypothesis was that a longer BP-limb in patients with higher BMI would result in more weight loss, so that the final attained BMI would be similar compared to patients with a lower preoperative BMI. The current study aimed to investigate the impact of various BP-limb lengths based on preoperative BMI in terms of weight loss and resolution of comorbidities.2 (n = 58), unknown BP-limb length (n = 2), and a BP-limb length of 250 cm (n = 11). All data were extracted from the electronic patient records. The medical ethical committee approved the study . All patients who underwent bariatric surgery had an operation indication in agreement with the international IFSO guidelines and provided written informed consent for the use of their data.This is a single-center retrospective cohort study of all patients who underwent a primary OAGB in a non-academic teaching hospital in the Northern Netherlands, from January 2015 to December 2016 (N = 744). Exclusion criteria were lost to follow-up (n = 29), pregnancy (n = 12), BMI > 50 kg/mPreoperative workup included multiple months of counseling provided by a dietician to prepare for the postoperative lifestyle regimen, including eating 6–8 times a day, separating eating and drinking, eating high-protein and healthy nutritional products, sufficient physical activity, and abstinence of alcohol and carbonated drinks. Candidates were screened by a multidisciplinary team evaluating mental health, detailed medical condition, and adherence to the postoperative lifestyle.2, a 180-cm BP-limb for BMI 40–44.9 kg/m2, and a 200-cm BP-limb for BMI 45–49.9 kg/m2. The ultimate BP-limb length was determined per-operative at the discretion of the surgeon and therefore could differ from the general rule. The small bowel was measured with a hand-over-hand technique, with 5 cm steps for measuring purposes. The standard instrument had a 4.5-cm metallic grasper and was used as a reference. All OAGB procedures were performed either by four experienced bariatric surgeons or by surgical residents under direct supervision of these bariatric surgeons.The surgical technique has been described before by Apers et al. . The BP-Weight loss surgery specific multivitamins and calcium/vitamin D supplementation were recommended lifelong. The patient’s follow-up visits took place after 1 month, 1 year (10–14 months), 2 years (18–30 months), and 3 years (30–42 months). Follow-up data were included until the 1st of June 2020. At each follow-up visit, standard care included inquiry after well-being and complaints, current medication use, side effects, evaluation of comorbidities, and measurement of body weight.2. The percent of total weight loss (%TWL) was defined as  / initial weight x 100. Remission of hypertension and diabetes mellitus type 2 (T2D) was defined as partial if the dosage of medication was decreased compared to the preoperative dosage. Total remission of hypertension and T2D was defined as discontinuation of the prescribed medication with normalization of the blood pressure or HbA1c.The percent of excess weight loss (%EWL) was defined as  /  x 100. Ideal weight was defined as the weight corresponding to a BMI of 25 kg/mData were presented as mean ± standard deviation, median [interquartile range], and number (percentages). Normally distributed values were compared using a t-test or one-way ANOVA, skewedly distributed variables were compared using the Mann-Whitney U test or Kruskal-Wallis test, and for categorical data, the Chi-squared test was used. A two-sided p-value of ≤ 0.05 was considered statistically significant. Bonferroni correction was applied when post hoc tests were performed.Multiple linear regression analyses were performed to analyze the effect of the different BP-limb lengths on %EWL and BMI loss when correcting for confounders. In both multiple regression analysis, preoperative BMI was selected as confounder. Statistical analyses were performed in SPSS version 24.2, respectively. Hypertension was documented in 209 (33%) and T2D in 126 (20%) patients. Late postoperative complications as ulcers were present in 3.6%, internal herniation in 0.6%, and cholelithiasis in 5% of the patients. Two patients died during the follow-up, not related to the OAGB procedure (0.3%). Follow-up percentages after 1, 2, and 3 years were 94, 86, and 74%, respectively.Baseline characteristics of the 632 patients are shown in Table 2 in both years , 180 cm in 388 61.4%), and 200 cm in 72 (11.4%) patients , the other groups showed no difference.After 3 years, attained BMI, BMI loss, and WL were significantly higher in groups with longer BP-limbs. %EWL was significantly higher in the 150-cm group compared to the other two groups, with no difference between patients in the groups of 180 and 200 cm. No difference in %TWL between the groups was found. As shown in Table After adjustment for preoperative BMI, a BP-limb length of 180 cm was significantly associated with 4,5% less %EWL at 1 year follow-up, compared to a BP-limb of 150 cm (P = 0.01) Table . After aThe results of this retrospective cohort study show that longer BP-limbs in patients with a preoperative higher BMI did not result in an attained BMI that was similar to the patients with a preoperative lower BMI. Furthermore, longer BP-limb lengths were not associated with higher BMI loss, after adjustment for the confounder preoperative BMI.Patients were selected for different BP-limb length based on their preoperative BMI. Our hypothesis was that a longer BP-limb in patients with higher BMI would result in more weight loss, so that final BMI in those patients would be similar to those with a preoperative lower BMI. The results of this retrospective cohort study do not support this hypothesis. The more BMI loss in the groups with the longer BP-limbs seems to be mainly caused by higher preoperative BMI and not by the longer length of the BP-limbs. This effect of preoperative BMI on weight loss outcomes corresponds with a retrospective cohort study on predictors of weight loss after RYGB in 2070 patients . Higher Using the percentage %EWL or %TWL as outcome when comparing different BMI groups introduces a bias . A higheRetrospective cohort studies on tailoring BP-limb based on BMI in OAGB show comparable results in terms of weight loss outcomes –19. ThesThe literature on the OAGB contains considerable variation in lengths used for the BP-limb. Some bariatric surgeons use a fixed length and some tailor the BP-limb based on patient-related parameters such as BMI, age, sex, diet, and comorbidities , 20–22. The length of the total small bowel can vary considerably, with measured values ranging from 350 to 1050 cm . The eff2 and 77%, respectively. These results are similar to other large series reported by bariatric centers who perform the OAGB [2 and %EWL 77 ± 24 %. This wide distribution of outcomes in BMI and %EWL are comparable with other large cohort studies in both OAGB and RYGB [2 in combination with a minimum of side effects. After 3 years of follow-up, 91% of the patients still had a BMI above 25 kg/m2. With the aims in mind, there is clearly room for improvement in bariatric procedures. Remission of hypertension and T2D both total and partial occurred in 90% and 99% of the patients, respectively. These results are comparable to other large cohort studies on OAGB [In our study population, satisfactory results of mean weight loss outcomes were seen. After 3 years of follow-up, BMI and %EWL were 29 kg/mthe OAGB –10, 21. and RYGB , 28. Wei on OAGB , 13, 21.This study emphasizes that further research is necessary to find the optimal way to determine the BP-limb length in the OAGB. The contribution of the total length of the small bowel to weight loss and nutritional deficiencies in OAGB needs further exploration. We have therefore started a randomized controlled trial comparing a fixed BP-limb length to one tailored based on total small bowel length looking at both achieving a healthy BMI with narrow range and few nutrient deficiencies .The strength of the present study lies in the large and representative population, with a relatively large number of participants in each group. The lost to follow-up over 3 years was 26%, which are realistic data from a bariatric center in the Netherlands. Limitations of this study were the retrospective study design and the lack of data on nutritional deficiencies and total small bowel length. As some studies found longer BP-limb lengths were associated with more nutritional deficiencies, this would have been a valuable addition to this study .In conclusion, in this retrospective study, tailoring BP-limb length according to baseline BMI did not abrogate the BMI differences at 3 years after OAGB with also no differences in resolution of comorbidities.


Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops—a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to “the proline rule”. Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min.A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium  Starch is a storage polysaccharide that is produced in plants as a result of the photosynthetic process. It is used directly as a food component and can be converted into plenty of biotechnologically important products by chemical or enzymatic treatment ,2. SpeciOligo-1,6-glucosidase  belongs to the subfamily 31 of the glycosyl hydrolase family 13 (GH13_31) . NumerouCold-active enzymes possess high catalytic activity at low temperatures as well as decreased thermal stability . TypicalExiguobacterium sibiricum (Exig_1739 in KEGG database) isolated from Siberian permafrost soil. This microorganism is known for its complex system of adaptation to permafrost conditions . T,6. TpNPGing site . The catB. cereus and B. coagulans as well as of the enzymes belonging to other families [According to the published data, the presence of proline residues in the loops, at the second positions of the β-turns, and at the N-termini of α-helices increases the rigidity of the protein molecule, thereby improving its thermostability. This so-called proline rule was successfully used to increase the thermal stability of oligo-1,6-glycosidases from families ,40,41. Ifamilies .To identify the target residues for mutagenesis, we have also used the alignment of EsOgl amino acid sequence with the sequences of homologous glucosidases . ConsequE. coli cells according to the protocol developed for the wild-type EsOgl. All isolated mutant proteins demonstrated high purity and electrophoretic mobility, corresponding to the calculated molecular weight of 66 kDa  and S130P (t1/2 29 min) that is 1.8- and 2.6-fold longer than that of the wild-type protein (11 min). The E176P mutant demonstrated only a moderate increase in the half-life at 45% and the half-life of the K441P (12 min) was almost the same as of the wild-type protein , ASE (t1/2 = 129 min) and ASEK (t1/2 = 88 min) mutants were significantly  increased, respectively, in comparison with the wild-type EsOgl  which are characteristic for this environmental niche [rmafrost . Accordial niche .E. sibiricum prefers sugars and carbohydrates as carbon sources and can degrade starch and glycogen. Consequently, numerous coding sequences for the enzymes which catalyze corresponding biochemical transformations were disclosed in the genome of this bacterium. The expression of several predicted glucosidases was found to be influenced by temperature including Exig_1739 and Exig_2537 genes encoding for the putative alpha-amylases [E. sibiricum genome contains genes encoding mesophilic and psychrophilic enzymes that can be differently expressed at changing temperature conditions. It was shown that amylases . Expressamylases . These dPsychrobacter cryohalolentis K5T [Lactobacillus plantarum [B. cereus, B. coagulans and B. thermoglucosidasius that possess bell-shaped temperature profiles of activity with a pronounced maximum [B. cereus (BceOgl)  .Pseudoalteromonas haloiplanktis (AHA), chitobiase from Arthrobacter sp. and many other cold-adapted enzymes [Arthrobacter sp., metalloprotease from Pseudomonas sp., and Atlantic cod uracil-DNA glycosylase, demonstrate an increased number of arginine residues in comparison with thermostable homologs. It was proposed that in this case, the arginine sidechains do not participate in stabilizing ionic interactions but rather interact with water molecules, thus increasing molecular flexibility [To obtain an explanation for this phenomenon, we have turned our attention to the unusual amino acid content of EsOgl . In comp enzymes . Howeverxibility .B. cereus and exceeds the number of prolines in the thermostable oligo-1,6-glucosidase from B. coagulans by four residues  were obtained, as well as three variants containing combinations of these mutations . Characterization of the recombinant proteins demonstrated significant differences between their properties and the properties of the wild-type enzyme . All mutB. cereus oligo-1,6-glucosidase for the prolines made a significant contribution to the protein stabilization. Moreover, the stepwise addition of the mutations demonstrated an additive effect on thermostability, i.e., provided a consistent increase in thermal stability. The most effective mutations appeared to be located at the second positions of beta-turns and the first positions of alpha-helices. The introduction of the proline residues into the loops led to a less significant stabilizing effect [Previously, it was shown that temperature adaptation of α-amylases can be provided without changes in the active site of the enzymes . Substitg effect .B. cereus [In the present work, we demonstrated that the introduction of the proline residues at the positions A109 (at the N-terminus of an α-helix) and S130 (in the loop region) resulted in the greatest increase of the thermal stability. Mutation of the homologous residue N109P also resulted in increased thermal stability of oligo-1,6-glucosidase from . cereus  and the Km values were concomitantly somewhat increased. However, considering a high substrate load routinely used in industrial applications, an increase in Km from 1 mM to 1.5 mM is not relevant. At the same time, the increased catalytic rate can be translated into lower enzyme consumption, shortened reaction time, or a smaller reactor size. Whereas increased thermostability may allow for a wider operational window and increased process stability of the enzyme. These findings confirmed the applicability of the proline rule approach for the cold-active oligo-1,6-glucosidases. However, its efficiency relies on a careful assessment of the individual contribution of each mutation for their subsequent combination in the target protein. In this work, we obtained several mutant variants of EsOgl, ranging from enhanced cold-active enzymes to typical mesophylic ones. These mutant enzymes represent prospective candidates for biotechnological use.In conclusion, we have demonstrated that the introduction of the additional proline residues into the EsOgl amino acid sequence not only contributed to a significant increase in thermostability, but also, in certain cases, led to an increased catalytic rate. The


The proportion of RKO apoptotic cells increased by interfering with the endogenous expression of NLK. We speculate that LncRNA XIST may upregulate NLK expression by downregulating miR-92b-3p, thereby promote the development of CRC. These results provide important information for the identification of novel potential targets for the prevention or treatment of CRC.The regulatory mechanism of NLK in the carcinomagenesis and progression of colorectal cancer (CRC) remains unclear. Here, we identified a single nucleotide polymorphism (SNP) site of NLK (rs2125846) as a new susceptibility locus for CRC risk located within an intron of the human NLK gene in a Chinese population. NLK downregulation led to a decrease in the ability of proliferation and migration of RKO cells  Colorectal cancer (CRC) is one of the most common malignant cancers worldwide. CRC treatments, including surgical resection, radiotherapy, and chemoradiotherapy, have improved in recent years. Statistics show that 50% of patients with stage III can be cured by surgery, 20% of patients will survive following adjuvant chemotherapy, and 30% of patients will relapse within 2-3 years. Therefore, only 20% of patients with stage III benefit from chemotherapy, leaving 80% exposed to unnecessary toxicity Approximately 35% of CRC risk can be attributed to genetic factors The genome sequencing project has revealed that more than 90% of genomes are transcribed into non-coding RNA (ncRNA), and only 2% of the human genome consists of protein-coding genes LncRNAs participate in the development of CRC by coordinating with miRNAs and protein-coding mRNAs. LncRNAs modify the expressions of targeted genes by competinging with miRNAs for the binding sites on the mRNAs. Recent studies have shown that regulatory networks formed by the LncRNA/miRNA/mRNA interactions regulate the initiation and development of CRC In this study, we explored the possible roles of NLK in the carcinomagenesis and progression of CRC from clinical perspectives with hope to provide meaningful implications for searching the markers for early diagnosis as well as drug targets for CRC treatment.147 CRC cases and 150 healthy subjects were enrolled in the present study. Healthy participants who matched the age and sex of the case group were randomly selected as the control group. Thirty-eight fresh CRC tissue pairs  were obtained by surgical resection from the First Affiliated Hospital of Anhui Medical University and immediately stored in RNAlater at -80 °C. All cases were diagnosed by histopathology, and patients with preoperative radiotherapy and chemotherapy or with autoimmune diseases such as diabetes and nephropathy were excluded. The tissue wax blocks, and sections of CRC were obtained from the Department of Pathology of the First Affiliated Hospital of Anhui Medical University and confirmed via histopathological diagnosis by pathologists. Informed consent was obtained from each enrolled patient. The study protocols were approved by the Ethics Committee of Anhui Medical University.DNA was extracted from peripheral blood leukocytes according to the manufacturer's instructions of the DNA extraction kit . The content and purity of DNA were detected using a NanoDrop One/OneC micronucleic acid protein concentration analyzer . The ratio of the absorbance at 260 and 290 nm was between 1.8 and 2.0.The high-resolution melting (HRM) method was used to determine the SNP genotypes. HRM statistics were collected from the Roche LightCycler96 fluorescence quantitative PCR instrument. The primers used were as follows: NLK-SNP-F, GCTCTAACTTCTAATGGAAGTC and NLK-SNP-R, CAGATCCATACATGAGAAACAC.Tissue sections were deparaffinized, rehydrated, and heated using citrate antigen repair solution under high pressure for antigen retrieval. Then, the slides were incubated with 3% hydrogen peroxide at room temperature to block endogenous peroxidase activity and subsequently incubated with the appropriate amount of a polyclonal anti-NLK antibody  for 3 hours at room temperature. The sides were washed three times with PBS, followed by incubation with the second antibody and and treated with DAB for 5 minutes. The slides were examined under a microscope, and the results were analyzed with Image J software to calculate the positive IHC signals.A one-step TUNEL apoptosis detection kit  was used to determine the rate of apoptosis in CRC tissues and adjacent normal tissues. After dewaxing in water, 20 μg/ml of DNase-free proteinase K was added for 30 minutes. The TUNEL detection solution (fluorescence labeling solution:TdT enzyme=9:1) was prepared and incubated with tissue sections for 30 minutes. The slides were washed three times with PBS, and DAPI dye solution was added for 3 minutes. After washing with PBS three times, an anti-fluorescence quencher was added dropwise. The slides were mounted and later observed under a microscope.The human CRC cell line RKO was purchased from the American Type Culture Collection (USA). It was then cultured in Dulbecco's Modified Eagle Medium  with 10% fetal bovine serum (FBS) and incubated in a humidified incubator with 5% CO2 at 37 °C. The NLK overexpression lentivirus vector NLK-LV5 (EF-1aF/GFP&Puro) and NLK interference lentivirus vector NLK-shRNA- LV3 (H1/GFP&Puro) were obtained from Shanghai Jima Pharmaceutical Technology Co. Ltd. For cell transfections, RKO cells were seeded into a 12-well plate, cultured overnight, and transfected with an appropriate amount of lentivirus containing NLK-LV5, negative control (LV5NC), NLK-shRNA-LV3, or negative control (LV3NC). The medium was replaced with fresh medium after 12 hours. The state of the cells and the proportion of GFP expression were observed daily. All transfects were tested regularly by qRT-PCR and western blot to ensure the efficiency of overexpression or knockdown. The target sequences of NLK shRNA were as follows: NLK-KD, 5'-GGATAGACCTATTGGATATGG-3' and LV3NC, 5'-TTCTCCGAACGTGTCACGT-3'.The proliferation rate of RKO cells was measured with the Cell Counting Kit-8  assay and colony formation assay. For the cell count assay, control and transfected cells were cultured in a 96-well plate (2000 cells/well) and incubated at 37 °C in 5% CO2. Triplicate wells were measured in each group. After adding 10 μl CCK-8 solution to each well, the plate was incubated for 2 hours. Then, the spectrophotometric absorbance was measured at a wavelength of 450 nm using a microplate reader  for each sample. For the colony formation assay, control and transfected cells were seeded into a six-well plate (400 cells/well) and incubated at 37 °C in 5% CO2 for 8-10 days. Triplicate wells were measured in each group. Cell culture was terminated when visible clones were observed, and then the colonies were stained with crystal violet. The colonies were carefully washed with ddH2O three times, and images were taken after drying. The number of colonies was counted three times, the average value was obtained, and the number of clones was compared among the groups.4 cells in 100 μl serum-free DMEM was added to the top chamber, and 600 μl of medium containing 10% FBS were placed into the bottom chamber as an attractant. The non-migrated cells were removed from the top chamber after incubation for 24 h. The chambers were then fixed with a 4% methanol solution, stained with crystal violet, and photographed.The cell migration assay was performed in a Transwell chamber . 5×10Flow cytometry was used to detect the apoptosis and cell cycle phase of CRC cells. Annexin V-PE and 7-ADD double staining  was performed in accordance with the manufacturer's instructions to detect cell apoptosis. Then, 200 μl 1X Annexin V Binding Buffer was added to the solution, and the cells were analyzed using a flow cytometer. For the cell cycle assay, RNase A and PI staining solutions were added to collected cells in accordance with the supplier's instructions . Then, the cell cycle was detected using a flow cytometer.Briefly, the harvested cells were washed with PBS and lysed with 1 X SDS lysis buffer. The BCA method was used to measure the protein concentration. Samples were separated by 10% SDS-PAGE and transferred to the PVDF membrane. The membranes were blocked with 5% fat-free milk in PBS, 0.1% Tween 20 for 2 hours at room temperature, followed by overnight incubation with the primary antibody at 4 °C. Next, the secondary antibody was incubated for 1 hour at room temperature, and the protein bands were detected using the ECL reaction solution.Total RNA was extracted from cultured cells using a total RNA extraction kit  in accordance with the manufacturer's instructions. Then, cDNA synthesis was performed using a reverse transcription kit  following the manufacturer's instructions. A fluorescent quantitative PCR kit  was used for qPCR analysis. The relative gene expression level was calculated by the 2ΔCt method. All experiments were repeated three times, and GAPDH was used as the internal control. The following primers were used: NLK: F: 5'-TATCGGGCTCCAGAAATCCT-3', and R: 5'-GTGTGCCCAACAGATCCGT-3'; LncRNA XIST: F: 5'-GAGGCAAGATGGATGATAGCAG-3', and R: 5'-ACAATCACGCAAAGCTCCTAAC-3'; miR-22-3p: 5'-CTGCTATTGCACTCGTCCC-3'; U6: F: 5'-CTCGCTTCGGCAGCACA-3', and R: 5'-AACGCTTCACGAATTTGCGT-3'.The 293T cells were cultured for co-transfections. The XIST and NLK 3′-UTR regions, containing potential miR-92b-3p binding sites, were predicted using TargetScan version 7.11. The predicted 3′-UTR fragments were amplified by PCR. Mutants were then constructed by introducing point mutations into the seed binding site for miR-92b-3p. The wild type and mutant fragments  were subcloned into the psiCHECK2 vector , downstream of the renilla luciferase gene. The vector also contains the firefly luciferase gene. Cells were seeded in 24-well plates and cotransfected with wild-type or mutated luciferase constructs along with miR-92b-3p mimics, miR-92b-3p inhibitors, or controls. The Dual Luciferase Reporter Assay System (Promega) was used 48 h after transfection following the manufacturer's protocol. The relative luciferase activity was calculated using the ratio of firefly luciferase activity to renilla luciferase activity.SPSS20.0 software was used for analysis. The results were subjected to one-way ANOVA and parametric t-testing and were expressed as the mean ± SD. An unconditional logistic regression model was used to analyze the association between different genetic models and CRC, and the odds ratio (OR) and 95% confidence interval (95%CI) were calculated. The AA genotype represents the homozygous wild type, the AG genotype represents the heterozygous mutation, and the GG genotype represents the homozygous mutation. The genetic models used in this study included a codominant genetic model , dominant genetic model [heterozygous + polymorphic homozygous vs wild-type homozygous--(GG+AG)/AA], and recessive genetic model [polymorphic homozygous vs wild-type homozygous + heterozygous --GG/(AA+AG)]. The goal of this analysis using the model referred is to determine risk factors for CRC. A logistic regression model was used to analyze the interaction between multiplicative models. Statistical significance was accepted at a level of P value < 0.05.This case-control study enrolled 147 CRC patients and 150 healthy subjects. The two groups were comparable and no significant difference in sex or age was found (Table th intron (2364A/G) of the NLK gene. An A-G base substitution was identified at position 2364 as follows, GAGAAACACTTTTATGATGTCTGCT[A/G]GTACCAGGGTAATTAAATCTCAA. The reverse mutation was A/G, and the forward mutation was T/C . In the case group, the AA genotype accounted for 44.1%, AG for 52.4%, and GG for 3.5% of the population, whereas in the control group, the figures were 46.7%, 42.0%, and 11.3% (P<0.05). Therefore, the rs2125846 polymorphism exhibited distinct patterns among the case group and the normal group (Table P>0.05) . Furthermore, the risk of CRC in patients with the GG genotype was 0.327 times that of the AA genotype. In the dominant genetic model, the AA genotype was considered the control group, and the corresponding P value for GG/AG was 0.654, indicating no significant difference. In the recessive genetic model, compared with the GG genotype, the G-A substitution that results in the AA/AG phenotypes was more closely associated with the onset of CRC (3.528-fold)  as opposed to tissue differentiation, tissue typing, age, tumor location, distant metastasis, TMN, and family history (P>0.05)   Figure C. The pr) Figure D.NLK knockdown in the CRC cell lines such as HT-29 and HCT116 affected cell proliferation, colony formation, migration ability, and led to cell cycle arrest at G1/S phase An ectopic copy of NLK was introduced into RKO cells using a lentivirus vector (NLK-LV5) with the empty vector (LV5 NC) as control. The RNA interference (RNAi) specifically targeting NLK expression was achieved with a lentivirus vector (NLK-shRNA-LV3). The overexpression (OE) lentiviral vector NLK-LV5 (EF-1aF/GFP&Puro) and the RNAi lentiviral vector NLK-shRNA-LV3 (H1/GFP&Puro) were used to infect RKO cells, and then the OE and RNAi stable cell lines were constructed by puromycin selection and flow sorting. The empty backbone vector was used as control for OE and a scrambled shRNA-containing vector as control for RNAi. Both qPCR and Western blot results confirmed the altered expressions of NLK as intended  to examine the apoptosis and cell cycle of RKO cells upon altering NLK expression. The size of apoptotic cell population was significantly reduced with NLK overexpression (OE) compared with the control, in contrast to the scenario in the NLK-deficient RKO cells where the apoptosis was greatly enhanced Figure A. Specifi Figure B. TherefP<0.05) (Figure There was an interesting pattern of differential expression among XIST, miR-92b-3p, and NLK emerging from the CRC clinical samples under the study. Specially, XIST exhibited higher expression in the CRC tissues than in the adjacent tissues whereases the opposite pattern appeared with miR-92b-3p P<0.0 (Figure i, the expression of miR-92b-3p was significantly upregulated while NLK expression was suppressed (Figure We then explored the functional relevance of this pattern to CRC in RKO cells. In response to XIST downregulation via RNAd Figure B. When td Figure C.We further tested whether interlinked expressions of XIST and miR-92b-3p or miR-92b-3p and NLK is mediated by direct interactions. We established a luciferase-based reporter system in which was to measure the influence of upstream stimulation on the transcription level of downstream target genes, that is, the influence on the mRNA expression level of target genes. First, miR-92b-3p mimics/NC and Report-XIST-wt/mut were co-transfected into 293T cells, the fluorescent intensity was measured for the expression of ectopic XIST 24 hours post transfection. As shown in Figure Next, we focused on the relationship between NLK and miR-92b-3p. Similarly, miR-92b-3p suppressed the expression of NLK, and disturbing the potential binding between two disrupted this regulatory relationship Figure B.Among all cancers, the incidence of CRC ranks third, and the mortality rate ranks second wordwide. In the last 10 years, the mortality and morbidity of CRC in China have increased In the past three decades, an increasing number of SNPs have been confirmed to be associated with CRC risk through extensive genome-wide association studies and candidate gene association studies Here, we identified a new susceptibility locus, rs2125846, for CRC risk located within an intron of the human NLK gene in a Chinese population. It was statistically related to the tumor size of CRC, the depth of CRC infiltration, and whether the patient had a history of alcohol consumption. We think there are two possible explanations for this association. First, the sample size was insufficient and mainly focused on the Han population in Anhui Province. However, more comprehensive CRC-related risk factors and common clinicopathological features shall be included along with enlarging sample sizes in future work.NLK has been proposed as a tumor suppressor of therapy interest We showed that NLK expression regulates the proliferation ability & migratory ability of RKO cells Figure . One aspLncRNAs participate in various biological processes by regulating gene expression at the transcriptional, post-transcriptional, and epigenetic levels MiR-92b was first reported to be overexpressed in primary brain tumors We demonstrated that XIST acts directly on the miR-92b-3p transcript and regulates its expression. In a similar way, miR-92b-3p regulates the expression of NLK. Overall data suggested a possible role ceRNA in CRC cells, in which the XIST/miR-92b-3p/NLK signaling axis could play critical roles, therefore possessing potential therapeutic values.


After mating, females continued to receive the aforementioned diets during gestation and lactation. Weaning occurred at 21-day-old Wistar rats and the male offspring were subdivided: control-control (C-C)—offspring of standard diet fed dams who received a standard diet after weaning (n = 9–11), control-HS (C-HS)—offspring of standard diet fed dams who received a HS diet after weaning (n = 9–11), HS-C—offspring of HS diet fed dams who received a standard diet after weaning (n = 9–11), and HS-HS—offspring of HS diet fed dams who received a HS diet after weaning (n = 9–11). At adulthood, the male offspring performed the elevated plus maze and open field tests. At 152-day-old Wistar rats, the offspring were euthanized and the amygdala was removed for redox state analysis. The HS-HS group showed higher locomotion and rearing frequency in the open field test. These results indicate that this group developed hyperactivity. The C-HS group had a higher ratio of entries and time spent in the open arms of the elevated plus maze test in addition to a higher head-dipping frequency. These results suggest less anxiety-like behaviors. In the analysis of the redox state, less activity of antioxidant enzymes and higher levels of the thiobarbituric acid reactive substances (TBARS) in the amygdala were shown in the amygdala of animals that received a high-salt diet regardless of the period (pre- or postweaning). In conclusion, the high-salt diet promoted hyperactivity when administered in the pre- and postweaning periods. In animals that received only in the postweaning period, the addition of salt induced a reduction in anxiety-like behaviors. Also, regardless of the period, salt provided amygdala oxidative stress, which may be linked to the observed behaviors.High-salt (HS) diets have recently been linked to oxidative stress in the brain, a fact that may be a precursor to behavioral changes, such as those involving anxiety-like behavior. However, to the best of our knowledge, no study has evaluated the amygdala redox status after consuming a HS diet in the pre- or postweaning periods. This study aimed to evaluate the amygdala redox status and anxiety-like behaviors in adulthood, after inclusion of HS diet in two periods: preconception, gestation, and lactation (preweaning); and only after weaning (postweaning). Initially, 18 females and 9 male Wistar rats received a standard ( Sodium chloride (NaCl), also known worldwide as salt, is one of the most widely used condiments in food processing . It is eIn addition to the known harmful health effects, the use of high-salt diets has recently been linked to cerebrovascular diseases and cognitive impairment in humans . StudiesOne of the main possible mechanisms for behavioral changes caused by salt consumption is related to the oxidative stress . EvidencTherefore, this study aimed to evaluate the effects of the high-salt diet on amygdala redox status and anxiety-like behaviors at adulthood, considering: (1) the inclusion of the salt in the preconception, gestation, and lactation periods (preweaning) and (2) the addition of salt in the diet only after weaning until adulthood (postweaning). The main hypothesis was that the high-salt diet may result in amygdala oxidative stress regardless of the period, which, in turn, would promote changes in anxiety-like behaviors at adulthood.Rattus norvegicus) were obtained from Laboratório de Pós-Graduação e Pesquisa (LPP-UFVJM) and housed in conditions of natural moisture, temperature of 22 ± 2°C (controlled by an air conditioner), and a 12-h cycle of light and darkness, with the light cycle beginning at 7:00 am. All the animals had free access to potable water and their respective diets.This experimental protocol was approved by the Ethics Committee on the Use of Animals of Universidade Federal dos Vales do Jequitinhonha e Mucuri (CEUA-UFVJM) (protocol 025/2018). These are also in agreement to the ethical principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). All the rats (Wistar—control (C): received standard diet   or high-salt (HS) diet: received laboratory chow with added salt  . Copulation was evaluated every morning and confirmed by the presence of sperm in the vaginal smear, which was considered the beginning of gestation. All the animals received food and water ad libitum. After this period, the nulliparous female rats (141 days old) were placed for mating with males  during the dark cycle (7:00 pm to 7:00 am) every day. Parents during mating  and dams during gestation and lactation continued receiving the aforementioned diets (control or HS). At birth, the litters were culled to eight pups .Initially, 18 female and 9 male Wistar rats aged 21 days were used. The animals were housed in 3 per box according to sex in order to randomly receive the diets for a duration of 120 days: ® CR-1) or HS diets . Therefore, the offspring were subdivided into the following groups: control-control (C-C)—offspring of standard diet fed dams who received a standard diet after weaning (n = 9–11), control-high-salt (C-HS)—offspring of standard diet fed dams who received a HS diet  after weaning (n = 9–11), HS-C—offspring of HS diet fed dams who received a standard diet after weaning (n = 9–11), and HS-HS—offspring of HS diet fed dams who received a HS diet  after weaning (n = 9–11).In the postweaning period, only male offspring were used, housed 3 animals per box. Male offspring was randomly allocated to receive either control , when behavioral tests were carried out. Approximately, 1–2 animals from each litter were used for the behavioral and redox status analyses, in order to reduce litter effects. The experimental design is shown in ®) was positioned above the arena and two independent, blinded, and experienced studies later evaluated the randomly arranged videos. Between the performances of the two behavioral tests (EPM and OF), the animals were kept with their respective diets in the conditions mentioned previously. All the equipment used was cleaned with 70% ethanol between each test to eliminate olfactory cues.All the tests were performed in an isolated room (130 lux) and in a double-blind manner. The offspring performed the elevated plus maze (EPM) (141 day-old) and open field (OF) (151 day-old) tests, both during the morning period (7:00–12:00 am). A camera  and submerged on cold (4°C) phosphate-buffered saline (PBS) , followed by the amygdala dissection . After, n at 4°C . Both th4) was used as standard and the results were reported as nM of FeSO4/mg protein  method . The ass protein .2PO4) and 1 mM diethylene-triamine-pentaacetic acid (DTPA) was added to the tissue homogenate. Following this, 0.2 mM of pyrogallol was added and its oxidation was measured at 420 nm for 250 s at interval of 10 s. The results were defined as one unit (U) of SOD per mg protein in the sample (U/mg protein) (For the activity of the antioxidant enzyme superoxide dismutase (SOD), a solution containing 50 mM potassium dihydrogen phosphate (KHprotein) .Catalase (CAT) activity was assessed by metabolizing hydrogen peroxide . To perf–1 × cm–1). One unit of GST activity was defined as the amount of the enzyme that catalyzed the formation of one μmol of product × min–1 × mL–1 (Glutathione S-transferase (GST) activity was estimated spectrophotometrically as previously described . The ass1 × mL–1 .The lipid peroxidation evaluation was performed using the thiobarbituric acid reactive substances (TBARS) method and is classified as an oxidative stress marker . A solutAll the redox analyses were performed in triplicate using a plate reader (UV/Visible U-200 L Spectrophotometer). Protein content was quantified using bovine serum albumin (BSA) (1 mg/ml) as the standard . The res®, Hamburg, Germany). Graphics were made using the GraphPad Prism® version 7.0 . Sample normality was evaluated using the Shapiro–Wilk test. Data with normal distribution were analyzed using the two-way ANOVA, with the factors: preweaning (received standard or HS diets until weaning) and postweaning (received standard or HS diets only after weaning). The Newman–Keuls was used as a post hoc test when appropriate (p < 0.05). Data with non-normal distributions were analyzed by the Kruskal–Wallis test with the Dunn’s post hoc test. Results are expressed as a mean and SEM.Statistical analysis was performed with Statistica software  = 9.19, p < 0.01]. The offspring who received a HS diet until weaning entered less in the open arms compared to the offspring of standard diet fed dams (p < 0.01). In addition, an interaction in the factors pre- and postweaning was observed . The C-HS group showed higher ratio of entries in the open arms compared to the C-C (p < 0.05), HS-C (p < 0.01), and HS-HS (p < 0.01) groups  = 4.36, p < 0.05]. The offspring who received a HS diet until weaning spent less time in the open arms compared to the offspring of standard diet fed dams (p < 0.05). Also, an interaction in the factors pre- and postweaning was observed . The C-HS group spent more time in the open arms in relation to the C-C (p < 0.05) and HS-HS (p < 0.05) groups  = 12.52, p < 0.01]. The offspring who received a HS diet until weaning showed lower head-dipping frequency compared to the offspring of standard diet fed dams (p < 0.01). Moreover, a difference in the interaction of pre- and postweaning factors was observed . The C-HS group performed head-dipping more frequently compared to the HS-C (p < 0.01) and HS-HS (p < 0.01) groups (p = 0.06) and grooming frequency (p = 0.73) in the EPM test  groups . Similar) groups . For the) groups . No diffEPM test .FPRE  = 5.12, p < 0.05]. The offspring who received a HS diet until weaning remained more time in the central zone of the OF test compared to the offspring of standard diet fed dams (p < 0.05)  = 16.59, p < 0.01]. The HS-HS group reported higher locomotion in relation to the C-C (p < 0.05), C-HS (p < 0.01), and HS-C (p < 0.01) groups  = 0.16, p < 0.05]. The C-HS and HS-C groups accomplished lower numbers of rearing in relation to the C-C and HS-HS groups (p < 0.05) (p = 0.27) and grooming frequency (p = 0.35) in the OF test  . The tot) groups . The rea < 0.05) . No diff OF test .FPOST  = 29.92, p < 0.001]. The offspring who received a HS diet after weaning reported less SOD activity compared to the offspring of standard diet fed dams (p < 0.001). In addition, an interaction in the pre- and postweaning diets was observed . The C-HS and HS-HS groups showed less SOD activity compared to the C-C and HS-C groups (p < 0.01)  = 5.69, p < 0.05]. The offspring who received a HS diet after weaning displayed less GST activity compared to the offspring of standard diet fed dams (p < 0.05). Also, an interaction in the pre- and postweaning diets was found . The HS-HS group reported less GST activity with respect to the C-C group  = 5.21, p < 0.05]. The offspring who received a HS diet after weaning reported higher TBARS compared to the offspring of standard diet fed dams (p < 0.05). Moreover, an interaction of pre- and postweaning diets was observed . The C-HS, HS-C, and HS-HS groups showed the higher TBARS levels compared to the C-C group (p < 0.05) (p = 0.16) and CAT (p = 0.57) evaluations  . For GST-C group . In the  < 0.05) . No diffluations .High-salt diets are consumed worldwide and are associated with cardiovascular morbidity and mortality. Noteworthy, HS intake has also been linked to behavioral changes in rodents. This study evaluated differential effects of HS and standard diet combinations given in the pre- or postweaning period. In this study, an increase in locomotion was showed in the group of animals that received a HS diet in, both, the pre- and postweaning period (HS-HS group). In addition, animals that received the HS diet only after weaning displayed a decrease in anxiety-like behaviors (C-HS group). Furthermore, both the groups showed amygdala oxidative stress, which may explain the behavioral changes observed.The HS-HS group received the HS diet in both the periods (pre- and postweaning) resulting in adulthood hyperactivity measured by higher locomotion in the OF test. Also, this group presented an increase in the rearing frequency, which can be classified as a vertical exploration, confirming a high activity . InteresCuriously, the spontaneously hypertensive rats (SHR) model consistently exhibits hyperactivity in the OF test . This moFurthermore, one of our main hypotheses was that a HS diet could promote changes in anxiety-like behavior at adulthood. In this study, the C-HS group reported less anxiety-like behavior in the EPM test, due to the higher ratio of entries and time spent in the open arms, in addition to the higher head-dipping frequency . Gilman It is well established that experiences of mother during preweaning periods can modify the developmental health trajectory of her offspring. However, in some cases, no significant deleterious effects are observed, as demonstrated in the EPM test by the groups that received a HS diet in the preweaning period (HS-C and HS-HS). These observations are combined with the Predictive Adaptive Response (PAR) hypothesis, which argues that some changes that occur in early life in response to aversive stimuli are important to provide an advantage later in life. The PAR hypothesis predicts that these changes occur through epigenetic programming, which may also bring specific costs in the adult environment, making the animal maladapted on certain occasions . HoweverReactive oxygen and nitrogen species can be considered as essential for the full development of neuronal functions when occurring in low or moderate amounts. However, at excessive levels, they are harmful and can lead to oxidative/nitrosative stress, causing damage to proteins, lipids, and nucleic acids . In turn2 consumption and modest antioxidant defenses . The radical ONOO– is a very powerful oxidant and nitrosating agent. Thus, besides generates a toxic molecule (ONOO–), this reaction decreases the NO availability. NO plays an important role as a vasodilator, thus reducing it also contributing to arterial hypertension  is a transcription factor that regulates the expression of several proteins, among them some involved in antioxidant defense system of cells. For example, antioxidant enzymes such as CAT, SOD, and GST are produced after activating the Nrf2 pathway . Previourtension . Notablyrtension .This study has some limitations. First, to better understand the relationship between amygdala oxidative stress and observed behavioral changes, it is necessary in the future the use of drugs that alter the production of reactive oxygen and nitrogen species and the evaluation of the Nrf2 expression. Second, the assessment of inflammation in the amygdala would be important to understand the real impact of a HS diet at the cellular level and the extent of tissue damage. Also, it is also important to evaluate serotonin levels in this brain region, since its concentration in the amygdala is directly related to anxiety-like behaviors. Third, the use of females is necessary, since sexual dimorphism is common in behavioral assessment studies. Females could have different responses due to other developmental vulnerabilities, altered neuroendocrine regulation, or placental and epigenetic different effects. Fourth, the evaluation of other tests related to anxiety-like (light-dark box and hole-board tests) and hyperactivity  behaviors must be performed to better understand the outcomes of this model. Fifth, finally, the next studies should assess blood pressure and heart rate, in an attempt to establish a link between these physiological responses and the observed behaviors.In summary, this study demonstrated negative effects of a HS diet on the amygdala redox state. In addition, a HS diet promoted hyperactivity when administered in the combination of pre- and postweaning periods and decreased anxiety-like behaviors when offered only in the postweaning period. To the best of our knowledge, this is the first study that indicates damage to the amygdala in addition to behavior changes, regardless of the period in which salt is added to the diet. This fact is highlighted, due to the large consumption of salt in the world , its relA HS diet promoted hyperactivity when administered in the pre- and postweaning periods. In animals that received only in the postweaning period, the addition of salt induced a reduction in anxiety-like behaviors. Regardless of the administration period, salt provided amygdala oxidative stress, which may be linked to the observed behaviors.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by Ethics Committee on the Use of Animals of Universidade Federal dos Vales do Jequitinhonha e Mucuri .All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Kicks-number recorded during FSKT-mult was lower in the control compared with RHIT1:6 (p = 0.001), RHIT1:7 (p < 0.001), RHITSSRT (p < 0.05), P1:7 (p < 0.001), and SSRT (p < 0.001). Kicking decrement index (DI) during FSKT-mult was lower after RHIT1:6 compared with control (p = 0.008), RHIT1:7 (p = 0.031), P 1:6 (p = 0.014), PSSRT (p = 0.041). (1) P1:7 and PSSRT can be used to improve taekwondo-specific agility and kicks-number, (2) RHIT1:6 is beneficial to maintain low DI, and (3) plyometric and different WRR-repeated-techniques can enhance kicks-number.The study assessed conditioning activities’ (CAs’) effects involving different work-to-rest ratios (WRR) on taekwondo athletes’ physical performance. Adolescent taekwondo athletes (age: 16 ± 1 years) randomly participated in the control six experimental conditions. Each condition was composed of standard warm-up and CA composed of plyometrics (P) or repeated high-intensity techniques (RHIT) performed using three different WRR: 1:6, 1:7, self-selected rest time (SSRT). After rest, athletes performed countermovement jump (CMJ), taekwondo specific agility test (TSAT), 10 s frequency speed kick test (FSKT-10s), multiple frequency speed kick test (FSKT-mult). P1:7, SSRT induced techniques higher number in FSKT-10s ( Taekwondo is an intermittent striking combat sport requiring kick techniques performed with high intensity, jumps, and changes of direction to control distance from the opponent during the preparation to deliver subsequent kicks . During In combat sports, conditioning activity with different procedures has been used in judo ,9, karatBoth taekwondo professionals and sport scientists aim to prepare athletes to better handle the competition demands. To do this, manipulation of different conditioning activities and work-to-rest ratios can be pursued to administer more effective training. To the current authors’ knowledge, no studies have investigated the combined effects of using plyometrics (P) or repeated high-intensity techniques (RHIT) with different work-to-rest ratios during warm-up sessions on subsequent specific taekwondo performances. For this reason, the present study aims to compare the effects of six different conditioning activities (P vs. RHIT) using different work-to-rest ratios  and a control condition on physical performances in young taekwondo athletes. Due to the beneficial effects of the specific technical work on the technical performance of taekwondo athletes , it was Following convenience sampling, twenty-seven taekwondo athletes  belonging to the same club volunteered to participate in this study. To participate in the study, all athletes had to meet the following inclusion criteria: (i) they should be young taekwondo athletes; (ii) they should have five years or more of taekwondo experience; (iii) they should present no history of disease, medication, or lower limb injuries or neuromuscular disorders; (iv) athletes should not be in a period of body mass reduction process; and (v) females could participate if they were in the follicular phase of their menstrual cycle during the experimentation. All participants regularly participated in competitions  for more than 2 years and trained 2 to 3 days per week (2 h per session). Athletes did not present any medical restrictions during the experimental period and refrained from any strenuous exercises 48 h before the beginning of the experimental sessions. Participants were given an in-depth explanation of the protocol before starting the study. After explaining the advantages and disadvantages of the study, parental consent was granted, and written informed consent was signed. The study was carried out in accordance with the Declarations of Helsinki and approved by a local research ethics committee (CPP SUD N° 0332/2021).This study used a randomized repeated-measures research design to investigate the effect of conditioning activities and work-to-rest ratios on countermovement jump test (CMJ), FSKT-10s, FSKT-mult, and taekwondo-specific agility test (TSAT) performances. Due to the study design, allocation concealment as well as blinding could not be executed, as the athletes were aware of the condition being performed.One week before the beginning of the experimentation, athletes were well familiarized with the experimental procedures. In order to overcome time-of-day effects, all testing sessions were conducted at the same time of day (5 p.m. to 7 p.m.). In a randomized fashion, taekwondo athletes were assessed using CMJ , FSKT-10The effort time  was chosen in an attempt to reflect the mean time of attacks and counter-attacks reported in taekwondo competition, and imposed ratios  were chosen as they represent the time-structure of taekwondo combat [For the evaluation of lower-limb jumping performance, the CMJ test was executed using an infrared jump system . Athletes started from a standing position and performed a rapid downward motion by flexing the knees and hips immediately followed by a rapid extension of these joints. To exclude the influence of arm swing, subjects were asked to keep their hands on their waists. No lower limb flexion or arm swing in the upward phase was allowed. Three trials were carried out, and the best performance was maintained for analysis. The intra-class correlation coefficient (ICC) for test–retest trial for the present study was 0.97.During the FSKT-10s test, each athlete must perform the maximum number of kicks against a bag by alternating the right and left lower limb. The technique used during the 10 s of test was the bandal-chagui. The total number of kicks represented the performance in this test was 15. The ICC for test–retest trial for the present study was 0.87.Each athlete performed five sets of FSKT-10s with a 10 s rest interval between repetitions . PerformThe athlete began the test in his/her fighting stance position and behind the start line. At his/her discretion, the athlete advanced to the center mark as fast as possible. Then, following his own choice, he turned towards partner 1 by a shift and performed a roundhouse kick with his lead leg. Then, he turned to the other side, shifted to partner 2, and performed another roundhouse kick with the other lead leg. Thereafter, he returned to the center, moved to partner 3 in guard position, and performed a double roundhouse kick. Finally, the athlete ran backward to the start/finish line . The perd) analysis was used to interpret the magnitude of differences between variables and classified according to Hopkins [d ≤ 0.2 , 0.2 < d ≤ 0.6 , 0.6 < d ≤ 1.2 (moderate), 1.2 < d ≤ 2.0 (large), 2.0 < d ≤ 4.0 (very large), and d > 4.0 (extremely large). The statistical significance level was set at p ≤ 0.05.Data were presented as mean and SD. The statistical analysis was performed using SPSS 20.0 statistical software . The normality of data sets was checked and confirmed using the Kolmogorov–Smirnov test. Sphericity was tested and confirmed using the Mauchly test. Data were analyzed using a one-way analysis of variance with repeated measurements to compare performances throughout different experimental conditions. Bonferroni was used as post hoc test. Standardized effect size (Cohen’s  Hopkins : d ≤ 0.2F6182 = 0.609 and p = 0.723). However, a significant difference in the number of kicks performed during the FSKT-10s was found (F6182 = 4.781 and p < 0.001) with the plyometric condition using 1:7 and SSRT ratios, which induced increased performances compared with the control condition , both p < 0.001, respectively). Similarly, agility performance differed across conditions (F6182 = 3.83 and p = 0.001) with the plyometric condition using 1:7 and SSRT ratios inducing improved agility  compared with the control condition , both p < 0.01, respectively). Concerning the total number of kicks recorded during the FSKT-mult, there was a condition effect (F6182 = 6.107 and p < 0.001) with lower values in the control condition compared with RHIT1:6  and p = 0.001), RHIT1:7  and p < 0.001), RHITSSRT  and p < 0.05), P1:7  and p < 0.001) and PSSRT conditions  and p < 0.001). Moreover, regarding the DI, there was a condition effect (F6182 = 3.079 and p = 0.007) with lower DI values recorded in the RHIT1:6 condition compared with the control ), RHIT1:7 ), P1:6 ratio ), and PSSRT conditions ).There was no difference in the CMJ height between conditions  using different work-to-rest ratios during such activities . The main findings of the present study were that conditioning activities did not affect muscle power, but plyometrics using 1:7 work-to-rest ratio resulted in a higher number of kicks executed in the FSKT-10s and increased agility in the TSAT compared with the control condition. Moreover, except for the P1:6 condition, the total number of kicks during the FSKT-mult was higher after all experimental conditions in comparison with the control condition. Furthermore, decrement index in the FSKT-mult was lower after RHIT1:6 compared with the control, RHIT1:7, P1:6, and P (SSRT) conditions.The absence of performance change in the CMJ observed in the present study is similar to that reported by other investigations ,15, whicThe 10 s frequency of the kick test is a practical tool that mimics many characteristics of taekwondo combat  and has been used to assess the anaerobic performance of taekwondo athletes ,15. In tFor taekwondo athletes, the FSKT-mult test seems to be a good tool to discriminate performance , as taekTo improve agility, PAP has been proposed as a relevant strategy in current scientific research . Due to The current study showed that the inclusion of repeated high-intensity techniques and vertical jumps using taekwondo-specific work-to-rest ratios in warm-up sessions were effective to reduce the kick decrement index, improve taekwondo-specific agility, and increase the number of techniques performed by taekwondo athletes in a 10 s specific test. Specifically, (1) plyometrics with 1:7 work-to-rest ratio or self-selected rest time can improve taekwondo-specific agility and kicks-number, (2) repeated high-intensity techniques with a 1:6 ratio are beneficial to maintaining a low kicking-decrement index, and (3) plyometric and repeated techniques with different work-to-rest ratios enhance kicks-number. These exercises can be adopted by coaches as a specific warm-up strategy before competitions because of their benefits as well as their low logistical demands. Coaches can also include these exercises in athletes’ daily warm-up regime to increase performance output during training sessions. Finally, it is also possible for coaches to modify the work-to-rest ratios and the rest interval between these activities and subsequent performance depending on the conditioning activity used in order to prepare athletes to manage the constraints of the competition. Based on the present study’s results, coaches and sport scientists in combat sports and other types of sports can use specific drills as conditioning activities during warm-up to induce post-activation performance enhancement. Moreover, organizing these activities by applying specific work-to-rest ratios seems to be a good strategy that should be investigated further.


The Kidney Failure Risk Equation (KFRE) predicts the 2- and 5-year risk of end-stage renal disease (ESRD) in patients with chronic kidney disease (CKD) stages 3a-5. Its predictive performance in advanced CKD and in specific disease aetiologies requires further exploration. This study validates the 4- and 8-variable KFREs in an advanced CKD population in the United Kingdom by evaluating discrimination, calibration and clinical utility.2 and < 15 ml/min/1.73m2 to guide further treatment.Patients enrolled in the Salford Kidney Study who were referred to the Advanced Kidney Care Service (AKCS) clinic at Salford Royal NHS Foundation Trust between 2011 and 2018 were included. The 4- and 8-variable KFREs were calculated on the first AKCS visit and the observed events of ESRD  within 2- and 5-years were the primary outcome. The area under the receiver operator characteristic curve (AUC) and calibration plots were used to evaluate discrimination and calibration respectively in the whole cohort and in specific disease aetiologies: diabetic nephropathy, hypertensive nephropathy, glomerulonephritis, autosomal dominant polycystic kidney disease (ADPKD) and other diseases. Clinical utility was assessed with decision curve analyses, comparing the net benefit of using the KFREs against estimated glomerular filtration rate (eGFR) cut-offs of < 20 ml/min/1.73mA total of 743 patients comprised the 2-year analysis and 613 patients were in the 5-year analysis. Discrimination was good in the whole cohort: the 4-variable KFRE had an AUC of 0.796  for predicting ESRD at 2-years and 0.773 (95% CI 0.736–0.810) at 5-years, and there was good-to-excellent discrimination across disease aetiologies. Calibration plots revealed underestimation of risk at 2-years and overestimation of risk at 5-years, especially in high-risk patients. There was, however, underestimation of risk in patients with ADPKD for all KFRE calculations. The predictive accuracy was similar between the 4- and 8-variable KFREs. Finally, compared to eGFR-based thresholds, the KFRE was the optimal tool to guide further care based on decision curve analyses.The 4- and 8-variable KFREs demonstrate adequate discrimination and calibration for predicting ESRD in an advanced CKD population and, importantly, can provide better clinical utility than using an eGFR-based strategy to inform decision-making.The online version contains supplementary material available at 10.1186/s12882-021-02402-1. Chronic kidney disease (CKD) is not a benign condition given worsening kidney function is an independent risk factor for progression to end-stage renal disease (ESRD), cardiovascular events and all-cause mortality . AccuratTo date, the Kidney Failure Risk Equation (KFRE) remains the most well-validated risk prediction tool, predicting the 2- and 5-year risk of progression to ESRD in patients with CKD stages 3a-5 .2 (interquartile range: 12-19 ml/min/1.73m2), irrespective of whether patients had diabetic nephropathy, hypertensive nephropathy, glomerulonephritis, autosomal dominant polycystic kidney disease (ADPKD) or other conditions. However, the authors did not validate the predictive performance of the 8-variable KFRE, which may be of particular relevance in advanced CKD given the potential prognostic importance for mineral-bone disease, acidosis and inflammation at CKD stages 4–5, and which are captured by the extra parameters of the 8-variable KFRE. Furthermore, whilst statistical measures of model performance were reported, such as discrimination and calibration, the clinical utility of the KFRE was not evaluated. However, measures of utility are recognised as a useful marker of prediction model performance  . PerfectCalibration, the extent the predicted risk scores accurately estimate the observed values, was visually assessed by a calibration plot. Here, the predicted risk scores are plotted against the observed outcome of ESRD, which is treated as a binary outcome, and a smoothing function is then applied . PerfectWhilst discrimination and calibration provide statistical measures of performance, both fail to adequately describe the clinical utility of a model. To address this, a decision curve analysis can be undertaken that illustrates the impact of a risk model in supporting decision-making at various threshold probabilities , 14. TheNet benefit, represented as true positive cases, can also be expressed as the number of unnecessary interventions avoided in a population by simply focusing on true negative cases. For our study, unnecessary interventions translate as identifying patients who would suit less intensive monitoring and for whom referral for transplantation or dialysis access planning could be delayed.2 and < 15 ml/min/1.73m2. In addition, the median time-to-ESRD was calculated for the optimal model to provide information on the appropriate timeframe for when dialysis access formation should be undertaken.When comparing different prediction methods, the model with the highest net benefit on the y-axis across the range of threshold probabilities would be deemed to be of optimal value . In thisSurvival curves for the 4-variable KFREs were produced to compare the differences in outcome between a death-censored analysis and an analysis in which death prior to ESRD is handled as a competing event.p-value of < 0.05 was considered statistically significant.All statistical analyses were conducted using R, version 4.0.2 . A The SKS received ethical approval from the North West Greater Manchester South Research Ethics Committee (REC15/NW/0818). Written informed consent was obtained from all patients. The methods described herein were carried out in accordance with relevant guidelines and regulations of the SKS.The reporting of this validation study complies with the TRIPOD  statement  Additio.2 (13-18 ml/min/1.73m2),A total of 743 patients were included in the 2-year analysis of the 4- and 8-variable KFREs Table . In thiswhich was similar across the disease categories, and the median uACR was 409 mg/g 85-1356 mg/g), which was comparatively higher in patients with glomerulonephritis and diabetic nephropathy than in patients with hypertensive nephropathy and ADPKD. All these characteristics were similar in the cohort of 613 patients, which comprised the 5-year analysis of the 4- and 8-variable KFREs  and 20% (95% CI 10–39%) respectively  and good discrimination was seen in the other disease categories except for ADPKD, which showed a much lower AUC of 0.600 (95% CI 0.328–0.872). These findings were similarly reproduced with the 8-variable 5-year calculations .When compared with an eGFR cut-off of < 15 ml/min/1.73m2. In addition, it was able to identify 15 more true positive cases per 100 patients and 15 extra true negative cases compared with using an eGFR of < 15 ml/min/1.73m2 to guide further treatment. The median time-to-ESRD for patients with a 5-year KFRE risk of ≥50% was approximately 20 months (10-37 months).At a 50% risk threshold, the 4-variable 5-year KFRE identified 14 extra patients per 100 who would progress to ESRD and could identify delaying intervention in 14 more patients per 100 when compared with using an eGFR< 20 ml/min/1.73mThe net benefit results of the 4-variable KFRE were similarly seen when using the 8-variable KFRE for 2- and 5-year risk prediction.The cumulative incidence of ESRD using the Kaplan-Meier survival curve, in which death prior to ESRD was censored, was compared to the cumulative incidence of ESRD when adjusted for death as a competing event. Using the 4-variable KFRE as the main example, Additional file This validation study shows that the use of the 4- and 8-variable KFREs can be of clinical utility in an advanced CKD population and offers evidence for switching towards a risk-based model of care above one that relies solely on eGFR thresholds to trigger intervention in high-risk patients.2 (12-19 ml/min/1.73m2) in their work closely matches the 16 ml/min/1.73m2 (13-18 ml/min/1.73m2) in ours and we share similar baseline patient characteristics of age and sex. The rates of ESRD were higher in their study compared to ours (42% and 64% reached ESRD at 2- and 5-years compared to 35% and 54% in our study), and this was reflected in higher KFRE risk scores. Nonetheless, we similarly found that patients with glomerulonephritis and diabetic nephropathy had the highest disease-specific risk scores whereas those with hypertensive nephropathy and ADPKD had the lowest. Our work extends upon the study by Hundemer et al. [We undertook a similar study to the one reported by Hundemer et al. , who recWe show for the first time that the 8-variable KFRE performs on par with its 4-variable counterpart for patients in the whole cohort and across disease aetiologies. The 8-variable KFRE has previously been shown to have a slightly better risk prediction compared to the 4-variable KFRE [We show that the 4-variable KFRE had good discrimination in the whole cohort for prediction of ESRD at 2- and 5-years with AUCs of 0.796 (95% CI 0.762–0.831) and 0.773 (95% CI 0.736–0.810) respectively. These were slightly lower than the AUC of 0.83 (95% CI 0.81–0.85) at 2-years and 0.81 (95% CI 0.77–0.84) at 5-years in the report by Hundemer et al. [With respect to calibration, we found the KFREs underestimated risk at 2-years and overestimated risk at 5-years, especially in patients with higher predicted risk scores. The overestimation of risk at 5-years is likely explained by the death-censored analysis, which was undertaken as per the original KFRE development study . We showIn contrast to the findings by Hundemer et al. , we high2 and < 15 ml/min/1.73m2 over a range of appropriate threshold probabilities. Specifically, the 2-year KFREs were superior at the 40% ESRD threshold and the 5-year KFREs were superior at the 50% ESRD threshold, both thresholds identified in the literature as being relevant to guiding further care [Our validation study offers novel insight into the clinical impact of the 4- and 8-variable KFREs in an advanced CKD population by assessing clinical utility through decision curve analyses, which incorporates the measures of discrimination and calibration . We showher care . This prWe consider that there are two important roles in the application of the KFREs in multidisciplinary advanced care clinics: risk communication and planning for RRT. Communicating risk to patients is important as it provides an avenue to engage, counsel and potentially modify behaviour for patients at high-risk. Using the KFRE has been shown to be far more accurate than subjectively determining patients’ risk: in a prospective study of 257 patients with CKD stages 3–5, the KFRE better matched 2-year outcomes of ESRD than the predicted estimates from nephrologists and patients, who both tended to overestimate risk .With accurate risk prediction comes the corollary of using thresholds to plan for RRT in a timely manner. Our AKCS clinic prioritises pre-emptive transplantation given that this affords the best long-term outcomes . Recogni2. This approach has provided significant cost-savings through the reallocation of resources to those most likely to progress to ESRD [In addition, appropriately timing the initial referral and triage into the AKCS clinic would also be valuable to maximise this treatment opportunity. Indeed, a proposed KFRE cut-off of ≥10% at 2-years has been reported to select patients into multidisciplinary advanced care clinics, a strategy that has captured high-risk patients with an eGFR> 30 ml/min/1.73m to ESRD  and was  to ESRD . FurtherA limiting step in the routine use of the KFRE at our institution is the need for conversion of uPCR to uACR and a change in practice would ideally be needed to help integrate an immediate and accessible risk score into our electronic patient record. It is also important to acknowledge that risk scores obtained by the KFRE should only be used along with clinical judgement given the complexities of care in advanced CKD, where shared decision-making regarding future RRT needs to take account of patients’ preferences, their comorbidities, symptoms and the competing risk of death prior to ESRD.Our study provides for the first time a comprehensive, independent, geographical validation of both the 4- and 8-variable KFREs in advanced CKD in a UK-based cohort with specific evaluation of discrimination, calibration and clinical utility. We also provide insight into the applicability of the KFRE in an advanced kidney care clinic setting by focussing attention on the importance of communicating risk to patients, facilitating pre-emptive transplant and planning for AV fistula formation. This will be of significance to institutions who are considering the merits of using the KFRE in their practices.There are important limitations to our work. Firstly, there may have been misclassification of patients with diabetic or hypertensive nephropathy as the majority of these patients had not undergone a renal biopsy. This, however, is reflective of routine practice where the clinical probability of these particular diseases typically outweighs the risk of undergoing a biopsy for diagnostic confirmation. Nonetheless, our patient characteristics are in keeping with what we would expect in specific disease aetiologies, notably with higher levels of albuminuria in diabetic nephropathy compared with hypertensive nephropathy. Secondly, we were dependent on converting uPCR to uACR for all our patients, which may have impacted the predicted risk scores, but the online conversion tool we used has been shown to be effective with KFRE calculations. Finally, our study cohort originated from a single-centre and was largely Caucasian, which limits the generalisability of our results to other diverse clinical settings.The KFRE is an accessible and useful tool for risk prediction in patients with advanced CKD and in different disease aetiologies. Based on its beneficial clinical utility, the KFRE could be used in multidisciplinary advanced kidney care clinics to help deliver personalised and accurate care. The communication of risk scores can help facilitate early discussion to optimise living donor pre-emptive transplant and assist in decisions on the timing for dialysis access formation. Its use is also likely to be beneficial when managing patients at earlier stages of CKD to identify those at risk of rapid progression. Prospective data would be welcome to highlight the effectiveness of the KFRE in these patient groups, which would help herald a paradigm shift towards the routine use of objective risk-based assessments in delivering optimal CKD care.Additional file 1. The 4- and 8-variable Kidney Failure Risk Equation calculations for the 2- and 5-year predicted risk of ESRD and the formula to convert uPCR to uACR.Additional file 2. TRIPOD checklist for reporting of validation studies.Additional file 3. Baseline characteristics of patients within the 5-year KFRE analysis.Additional file 4. Comparison of the SKS study cohort to the KFRE development cohort.Additional file 5. AUC comparison between disease aetiologies for the 4- and 8- variable KFRE.Additional file 6. Tabulated overall calibration for 4- and 8-variable KFRE according to disease aetiology.Additional file 7. Sensitivity analysis to show probability of events with 1-Kaplan Meier estimate (death as a censored event) compared with cumulative incidence function (death as a competing event).Additional file 8. Raw dataset to perform the analysis.


However, due to the lack of standardized definition of Cas trans-cleavage enzymatic units, it is difficult to standardize the present CRISPR-Dx systems, which have undoubtedly impeded the development of the CRISPR-Dx industry. To solve the problem, we here first systematically optimized the reaction systems for Cas12a, and then defined its trans-cleavage units (transU), which we believe will be of great importance and interest to researchers in both molecular diagnostic industry and basic research. Moreover, a simple protocol was provided to facilitate a step-by-step measurement of the Cas12a transU, which can also act as a reference for the definition of the transU for other Cas proteins.The CRISPR diagnostic (CRISPR-Dx) technology that employs the  The tra (ssDNA) . Under tactivity . Similar targets . With thntatives .trans-cleavage activities, illuminating the fluorescent signals. HOLMES detects target nucleic acid sequences with the attomolar sensitivity and distinguishes single-base mismatches  and supports one-pot diagnosis of target nucleic acids  (HOLMES first uses the polymerase chain reaction (PCR) technology to amplify the target nucleic acids and then detects the amplicons with the Cas12a smatches . Howeveric acids . Unlike LOCKing)  were devLOCKing) . RecentlLOCKing) .In comparison with traditional molecular diagnostic methods such as polymerase chain reaction (PCR), CRISPR-Dx methods have shown great advantages in sensitivity, specificity, simplicity and portability and have been well recognized as the next-generation diagnostic methods . Therefotrans-cleavage units (transU), the Cas enzymes used in present systems are usually quantitated in concentration instead of enzymatic units. As the Cas specific trans-cleavage activities may vary from different commercial providers, distinct diagnostic performances can be obtained for a defined CRISPR-Dx system if the Cas enzyme is definitized in concentration, which will undoubtedly limit large-scale industrial applications of CRISPR-Dx technologies. Besides, it is well known that enzymatic cleavage activities can be affected by many reaction factors, including ions, salt concentration, pH values and temperature. Therefore, we here systematically studied the factors affecting Cas12a trans-cleavage activities, optimized the reaction systems and finally defined its transU. Moreover, a protocol was provided to simplify the transU definition procedures.However, due to the lack of unified trans-cleavage activity, the ssDNA reporter used was dual labeled with fluorophore and quencher , and the reaction was monitored by a fluorescence reader. To simplify the calculation, the trans-cleavage activity was calculated on the basis of the initial fluorescence growth rate vg (vg = ΔF/Δt), following the recently published Michaelis-Menten model aminomethane (abbreviated as Tris) salt in buffer F. Among the tested conditions, Cas12a generally preferred buffers with high pH values and exhibited obviously higher trans-cleavage activities in the 1 M glycine buffer (pH 8.6) and the 1 M Tris–HCl buffer (pH 8.5)  was finally chosen. Meanwhile, the loss of Cas12a trans-cleavage activities in buffer C could be caused by either the relatively low pH value or the low concentration of Mg2+, which has been demonstrated to be important for the maintenance of Cas12a cis-cleavage activities in a previous study  . Consideus study .trans-cleavage activities, and several components such as polyethylene glycol (PEG) and glycerol obviously enhanced the cleavage signals and were chosen for further analysis  and the concentrations of the FQ-reporter, including both un-cleaved  and the cleaved  reporters  was defined as the amount of Cas12a that trans-cleaves 1 pmol 8C FQ-reporter in 1 min at 37°C in a total reaction volume of 20 μL in HOLMES Buffer 1. To simplify the transU measurement, a step-by-step protocol was provided in the trans-cleavage efficiencies can be remarkably affected by the crRNA and target dsDNA sequences as well as their concentrations, the transU should be determined using the same sequences and concentrations as provided in this study to unify the measurement   .trans-cleavage reaction conditions, measured the Cas12a enzymatic constants and defined its transU, which will undoubtedly facilitate the development and application of robust Cas12a-based CRISPR-Dx systems in the future. Moreover, following a similar approach, the transU of other Cas effectors such as Cas12b, Cas12f, and Cas13 can be defined and their measurement protocols can be easily developed. On the other hand, although Cas12a exhibits much better performance in HOLMES Buffer 1 , one should be aware that there is still much work to do to further improve the Cas12a trans-cleavage activities, which may include the optimization of not only the reaction buffers but also the crRNA sequences in the future.Taken together, we here systematically optimized the Cas12a pH slice plate (Cat. No. #HR2-070) were ordered from Hampton Research .LbCas12a (LbCpf1) (Cat. No. #32108-03) was ordered from Tolo Biotech. . crRNAs were chemically synthesized by Biolino Acid Technology . FQ-labeled ssDNA reporters were ordered from Sangon Biotech. . The additive screening plates (Cat. No. #HR2-072) and the For the short dsDNA target, two complementary oligonucleotides were synthesized by commercial companies and then annealed to form dsDNA. Specifically, a pair of oligonucleotides (4 μM each) were mixed in 1× PCR buffer and then annealed in the following procedure with a thermal cycler: 95°C for 5 min and then reduced from 95 to 20°C at a rate of 1°C per minute. The annealed dsDNA target was diluted to 1 μM and stored at −20°C before use.trans-cleavage reaction was performed in a 20-μL volume, following the conditions as described before (vg) was calculated according to the maximum slope of the fluorescence curve using the ICEKAT software1.The Cas12a d before . In detav0 = vmax [S]/(Km + [S]), where [S] is the substrate concentration, vmax is the maximum reaction rate, and Km is the Michaelis constant. The turnover number (Kcat) was determined by the equation: Kcat = vmax/[Et], where Et was the effective complex. The concentration of the activated Cas12a enzymatic complex was always kept at 1 nM in 1× reaction buffer. The Cas12a trans-cleavage reaction was initiated by the addition of the ssDNA FQ-reporter at the final concentrations of 0 nM, 3.9 nM, 7.8 nM, 15.6 nM, 31.2 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1 μM, respectively. Reactions were carried out in three replicates at 37°C and fluorescence readouts were taken every 15 s. Background-subtracted fluorescence signals were obtained by subtracting the signal from a negative control. The v0 data were converted to nM/s from Relative Fluorescent Units using the calibrated standard curve (ref to section “6.1” in v0 against the substrate concentration and then fitting into the Michaelis-Menten curve (Graphpad Software).The Michaelis-Menten was analyzed according to following equation: The original contributions presented in the study are included in the article/trans-cleavage units. HL and JinW drafted the manuscript. DG, HZ, YX, and JinW analyzed the data, revised the manuscript, and supervised the study. All authors contributed to the article and approved the submitted version.HL, JianW, and JZ performed most of the experiments. YC, LY, and DJ provided assistances in defining the Cas12a LY is employed by Tolo Biotechnology Company Limited, Shanghai, China. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.